Biologically active human blood clotting Factor X is fully gamma carboxylated. Factor X, a vitamin K-dependent two-chain glycoprotein, is a substrate for both the extrinsic (tissue factor/FVIIa) and intrinsic (FVIIIa/FIXa) tenase complexes thus linking these two pathways (Kalafatis et al.,1994, Biochem. Biophys. Acta 1227:113). The activated form of Factor X (FXa) is the serine protease component of the enzymatic complex termed prothrombinase, the only known physiological activator of prothrombin. Prothrombinase assembles through reversible interactions between FXa and the cofactor factor Va (FVa) on an appropriate membrane (i.e., platelet) surface in the presence of Ca2+ ions (Mann et al., 990, Blood 76: 1). While FXa catalyzes prothrombin activation, the macromolecular interactions which stabilize prothrombinase lead to a substantial enhancement in catalytic efficiency (Mann et al.,1988, Ann. Rev. Biochem. 57:915), indicating that assembly of this complex is an important requisite for rapid and localized thrombin generation. Because Factor X/FXa occupies a central position in the coagulation pathway, there is considerable interest in its therapeutic modulation (Hauptmann et al.,1999, Thromb. Res. 93:203), highlighting the need to better understand structural determinants on Factor X/FXa important to its function.
While extensive progress has been made in delineating structural determinants important for function on thrombin, FIXa, FVIIa, and activated protein C (APC), less is known about FXa. One explanation is the limited number of naturally occurring FXa mutations to study. Another reason is the difficulty in producing (as compared to other vitamin K-dependent proteins) functional recombinant Factor X/FXa (rFXa). As with all vitamin K-dependent proteins, the biosynthesis of Factor X is complex, involving several co- and post-translational modifications (Kaufman R J, 1998, Thromb. Haemost. 79:1068). Efficient processing and release of mature two-chain Factor X into the circulation requires, 1) removal of the signal sequence, 2) formation of disulfide bonds, 3) modification of amino-terminal glutamic acid residues to γ-carboxyglutamic acid, 4) modification of one aspartic acid in the first epidermal growth factor (EGF) domain to β-hydroxyaspartic acid, 5) addition of N- and O-linked oligosaccharides to the activation peptide, 6) removal of an internal tripeptide to yield two chain Factor X, and 7) removal of the propeptide just prior to secretion (for review see Kaufman R J, 1998, Thromb. Haemost. 79:1068). While some of these modifications do not appear essential for Factor X function, the removal of the signal sequence, propeptide, internal tripeptide, and full γ-carboxylation are all steps which are important requisites for the production of biologically active Factor X/FXa.
Expression of rFactor X is heterogeneous with respect to removal of the internal tripeptide, propeptide cleavage, and γ-carboxylation. Expression of rFactor X/FXa in CHO and COS-1 cells appears less efficient than HEK 293 cells with respect to these modifications (Messier et al., 1991, Gene 99:291; Wolf et al., 1991, J. Biol. Chem. 266:13726; Rudolph et al., 1997, Protein Expression and Purification 10:373; Sinha et al., 1994, Thromb. Res. 75:427; Larson et al., 1998, Biochemistry 37:5029). Some of these inefficient modifications can be overcome by expressing rFactor X in HEK 293 cells, cotransfecting with PACE/furin, and modifying the Factor X propeptide at position −2 (Thr→Arg; henceforth referred to as native rwtFactor X). However, inefficient γ-carboxylation still remains a major problem (Rudolph et al., 1997, Protein Expression and Purification 10:373; Larson et al., 1998, Biochemistry 37:5029). For example, it has been discovered that on average only 32% of the rFactor X produced by HEK 293 cells is fully γ-carboxylated while the remaining material exhibits no γ-carboxylation (Larson et al., 1998, Biochemistry 37:5029). While separation of uncarboxylated and fully γ-carboxylated rFactor X can be readily accomplished, the resulting protein yields are less than desirable. This heterogeneity in γ-carboxylation can be overcome completely by expressing Gla-domainless rFactor X (Rezaie et al., 1993, J Biol. Chem. 268:8176); however, this is a less than satisfactory solution for studies involving macromolecular complex assembly of Factor X/FXa which requires a membrane surface. Thus, an ideal expression system would direct high-level protein production (>2–5 μg rFactor X/106 cells/24 hour) while still allowing for efficient execution of post-translational modifications essential to Factor X/FXa function.
The enzyme responsible for modification of glutamic acid residues to γ-carboxyglutamic acid (Gla) in the amino-terminal portion of a number of blood coagulation proteins is the vitamin K-dependent γ-glutamyl carboxylase (Wright et al., 1995, Vitamin K-Dependent g-Glutamyl Carboxylase, in High KA, Roberts H R (eds): Molecular Basis of Thrombosis and Hemostasis, New York, Marcel Dekker, Inc., p 309). The mechanism by which the carboxylase recognizes its substrate is believed to be through initial binding to an 18 amino acid propeptide sequence on the vitamin K-dependent protein (for review see Furie et al., 1990, Blood 75:1753). The importance of the propeptide sequence for γ-carboxylation is demonstrated by studies which show that disruption of this site in FIX, protein C, or prothrombin yield a mature protein that either lacks or is deficient in γ-carboxylation (Jorgensen et al., 1987, Cell 48:185; Foster et al., 1987, Biochemistry 26:7003, Furie et al., 1990, Blood 75:1753), indicating that the propeptide is required for γ-carboxylation. Analysis of naturally occurring mutations in this region supports this conclusion (Chu et al., 1996, J. Clin. Invest. 98:1619; Stanley et al., 1999, Biochemistry 38:15681). Recent studies also support the notion that the γ-carboxylation recognition site on the propeptide is sufficient to direct γ-carboxylation of glutamic acid residues as long as these residues are within 40 amino acids of the γ-carboxylation recognition site (Furie et al., 1997, J. Biol. Chem. 272:28258).
As noted above, in order that Factor X is biologically active, it must be fully gamma-carboxylated. Until the present invention, it has only been possible to produce biologically active rFactor X which is about 20–40% gamma carboxylated (Larson et al., 1998, Biochemistry 37:5029–5038). There is thus a great need in the art for methods of producing rFactor X which is fully carboxylated. In addition, there is also a great need for the development of methods of producing other mature vitamin K-dependent proteins that are fully gamma carboxylated. The present invention satisfies these needs.